Monoazo dyes with cyclic amine as fluorescence quenchers

ABSTRACT

The present disclosure provides reactive quencher dyes that can be used in the detection and/or quantification of desirable target molecules, such as proteins, nucleic acids and various cellular organelles. These dyes are essentially non-fluorescent but are efficient quenchers of various fluorescent dyes. Also, provided are methods of using the dyes, bio-probes incorporating dyes and methods of using the bio-probes. The quencher dyes described herein are modified to provide beneficial properties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 2, 2018, isnamed ENZ-111-SL_ST25.txt and is 817 bytes in size.

1. BACKGROUND

Current methods for detecting and/or quantifying nucleic acids ofinterest in clinical samples include nucleic acid amplification andreal-time detection. See. e.g., U.S. Pat. Nos. 5,994,056 and 6,174,670(measuring enhanced fluorescence of intercalating agents bound todouble-stranded nucleic acids); and U.S. Pat. Nos. 5,455,175 and6,174,670 (real time measurements carried out during the course of thereaction using a PCR cycler machine equipped with a fluorescencedetection system and capillary tubes for the reactions). In thesemethods, as the amount of double-stranded material increases duringamplification, the amount of signal also increases. Accordingly, thesensitivity of these systems depends upon a sufficient amount ofdouble-stranded nucleic acid being produced to generate a signal that isdistinguishable from background fluorescence. A variation of this systemuses PCR primers modified with quenchers that reduce signal generationof fluorescent intercalators bound to a primer dimer molecule. See,e.g., U.S. Pat. No. 6,323,337.

Another method of detecting and/or quantifying nucleic acids of interestincludes incorporation of fluorescent labels. See, e.g., U.S. Pat. No.5,866,336. In this system, signal generation is dependent upon theincorporation of primers into double-stranded amplification products.The primers are designed such that they have extra sequences added ontotheir 5′ ends. In the absence of a complementary target molecule, theprimers adopt stem-loop structures through intramolecular hybridizationthat bring a fluorescence resonance energy transfer (FRET) quencher intoproximity with an energy donor, thereby preventing fluorescence.However, when a primer becomes incorporated into double-strandedamplicons, the quencher and donor are physically separated and the donorproduces a fluorescent signal. The specificity of this system dependsupon the specificity of the amplification reaction itself. Since thestem-loop sequences are derived from extra sequences, the T_(m) profileof signal generation is the same whether the amplicons were derived fromthe appropriate target molecules or from non-target sequences.

In addition to incorporation-based assays, probe-based systems can alsobe used for real-time analysis. For instance, a dual probe system can beused in a homogeneous assay to detect the presence of appropriate targetsequences. In this method, one probe comprises an energy donor and theother probe comprises an energy acceptor. See European patentapplication publication no. 0 070 685. Thus, when the target sequence ispresent, the two probes can bind to adjacent sequences and energytransfer will take place. In the absence of target sequences, the probesremain unbound and no energy transfer takes place. Even if by chancethere are non-target sequences in a sample that are sufficientlyhomologous that binding of one or both probes takes place, no signal isgenerated since energy transfer requires that both probes bind in aparticular proximity to each other. See U.S. Pat. No. 6,174,670. Theprimer annealing step during each individual cycle can also allow thesimultaneous binding of each probe to target sequences providing anassessment of the presence and amount of the target sequences. In afurther refinement of this method, one of the primers comprises anenergy transfer element and a single energy transfer probe is used.Labeled probes have also been used in conjunction with fluorescentintercalators, which combines the specificity of the probe methodologywith the enhancement of fluorescence derived from binding to nucleicacids. See e.g., U.S. Pat. No. 4,868,103 and PCT Publication no. WO99/28500.

Other types of probes used in real-time detection and/or quantificationof nucleic acids of interest include an energy donor and an energyacceptor in the same nucleic acid. In assays employing these probes, theenergy acceptor “quenches” fluorescent energy emission in the absence ofcomplementary targets. See, e.g., U.S. Pat. No. 5,118,801 (“molecularbeacons” used where the energy donor and the quencher are kept inproximity by secondary structures formed by internal base pairing). Whentarget sequences are present, complementary sequences in the molecularbeacons linearize by hybridizing to the target, thereby separating thedonor and the acceptor such that the acceptor no longer quenches theemission of the donor, which produces signal. In Taqman, use is made ofthe double-stranded selectivity of the exonuclease activity of Taqpolymerase. See U.S. Pat. No. 5,210,015. When target molecules arepresent, hybridization of the probe to complementary sequences convertsthe single-stranded probe into a substrate for the exonuclease.Degradation of the probe separates an energy transfer donor from thequencher, thereby releasing light from the donor. See U.S. PatentPublication no. 2005/0137388 (describing various formats for utilizationof FRET interactions in various nucleic acid assays).

Probes comprising a non-fluorescent dark dye as energy acceptor(quencher) have also been used in the methods described above. When inclose proximity, quenchers absorb emitted fluorescence from a donor dyeand give no emission. Dabcyl is one such quencher with manyapplications, but its short absorption wavelength limits its use only tofluorescent reporters with short emission wavelengths, such asfluorescein and coumarin dyes. See, e.g., U.S. Pat. Nos. 5,866,336,5,919,630, 5,925,517, and 6,150,097 and PCT publication nos.WO9513399A1, WO9929905A2, WO9949293A2, WO9963112A2. Dark quenchers thatare suitable for pairing with long wavelength (red) fluorescent dyeshave also been developed, but they generally have more complexstructures, such as bisazo dyes (U.S. Pat. Nos. 7,019,129; 7,109,312;7,582,432; 7,879,986; 8,410,255 and 8,440,399; and PCT publication no.WO2014021680), azo dyes containing nitro-substituted naphthalene moiety(U.S. Pat. Nos. 7,439,341 and 7,476,735), azo dyes containing1,3,3-trimethyl-2-methyleneindoline ring system (U.S. Pat. No.7,956,169), nitro-substituted non-fluorescent asymmetric cyanine dyes(U.S. Pat. Nos. 6,080,868 and 6,348,596), N-aryl substituted xanthenedyes (U.S. Pat. No. 6,323,337), dyes containing anthraquinone moieties(U.S. Pat. No. 7,504,495), and azo dyes containing heterocyclic moieties(US publication nos. 2010/0311184, and DE 102005050833 and DE102005050834). Accordingly, there is a need for dark quencher dyes thathave simple, non-complex structures that are able to absorb and quenchfluorescence in a wider wavelength range.

2. SUMMARY

The present disclosure provides a series of fluorescence quenchers thatare monoazo dyes comprising cyclic amine groups. The monoazo dyesdescribed herein are essentially non-fluorescent in nature but areefficient quenchers of dyes that emit fluorescence over a larger rangeof wavelengths as compared to known quenchers. In particularembodiments, the quencher dyes described herein quench fluorescenceemission over wavelengths from about 500 nm to about 700 nm. As usedherein, the terms “quencher,” “dark quencher,” and “non-fluorescent darkdye” refer interchangeably to the monoazo dyes described herein thathave the ability to suppress emission of fluorescence from a donor dyeand that do not emit the absorbed fluorescence.

The present disclosure also provides a composition comprising a moleculeattached to a monoazo dye described herein. In some embodiments, themolecule is a nucleic acid. In other embodiments, the molecule is aprotein. In some embodiments, the monoazo dye is modified by theaddition of a reactive group (R_(x)). In other embodiments, the moleculeis modified by the addition of a reactive group.

Also provided are processes for qualitatively or quantitativelydetecting the presence of a single stranded or double-stranded nucleicacid of interest in a sample using a dark quencher compound describedherein.

The present disclosure additionally provides compositions for detectingvarious nucleic acid targets by techniques comprising but not limited toquantitative PCR and flow cytometry. In various embodiments, thecomposition is a kit comprising in packaged combination: (a) one or moremonoazo dye compounds described herein or a molecule covalently attachedto one or more monoazo dye compounds described herein; and (b)instructions for their use. In certain embodiments, the monoazo dye ismodified by the addition of a reactive group.

The disclosure further provides a composition comprising a solid supportto which is covalently or non-covalently attached one or more of themonoazo dye compounds described herein, wherein the compound orcompounds are modified by the addition of a reactive group (R_(x)) forattachment of the dye to a target molecule.

In other embodiments, the monoazo dyes described herein are utilized asa component of one or more probes for use in a multiplex assay fordetecting and/or quantifying one or more species of interest in amixture, such as a biological sample. In a typical multiplex assay twoor more distinct species are detected using the a monoazo compounddescribed herein and probes labeled with a donor fluorophore. In theseassays preferred species rely on donor-acceptor energy transfer suchthat the fluorescent species is bright and spectrally well-resolved andthe energy transfer between the fluorescent species and the monoazoquencher is efficient.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application to mean oneor more unless the context clearly dictates otherwise. Further, the term“or” is used in the present application to mean the disjunctive “or” orthe conjunctive “and.”

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like that has been included in this specification issolely for the purpose of providing a context for the presentdisclosure. It is not to be taken as an admission that any or all ofthese matters form part of the prior art or were common generalknowledge in the field relevant to the present disclosure as it existedanywhere before the priority date of this application.

The features and advantages of the disclosure will become furtherapparent from the following detailed description of embodiments thereof.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the UV-VIS spectrum of Compound 5.

FIG. 2 shows the UV-VIS spectrum of Compound 8.

FIG. 3 shows the UV-VIS spectrum of Compound 10.

FIG. 4 shows the UV-VIS spectrum of Compound 13.

FIG. 5 shows the UV-VIS spectrum of Compound 15.

FIG. 6 shows the UV-VIS spectrum of Compound 22.

FIG. 7 shows fluorescent trace of amplification (qPCR assay) using Cy5dye with Compound 23.

FIG. 8 shows fluorescent trace of amplification (qPCR assay) usingFluorescein with Compound 6.

FIG. 9 shows fluorescent trace of amplification (qPCR assay) usingRed598s (Enzo Life Sciences, Inc. Farmingdale, N.Y.) with Compound 6.

FIG. 10 shows a Flow Cytometry data of E6/E7 negative Pap smear sampleusing molecular beacon consisting of Fluorescein and Compound 6.

FIG. 11 shows a Flow Cytometry data of E6/E7 positive Pap smear sampleusing molecular beacon consisting of Fluorescein and Compound 6.

4. DETAILED DESCRIPTION

In certain embodiments, the disclosure is directed to a monoazo dyehaving the structure of Formula I:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are each independentlyselected from H, F, Cl, Br, I, CN, nitro, azido, hydroxyl, amino,hydrazino, aryl, substituted aryl, aroxyl, substituted aroxyl, alkenyl,alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino,diarylamino, alkyl(aryl)amino, alkanoylamino, alkylthio, alkylcarbonyl,aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkyloxycarbonyl,aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl,arylcarboxamido, or Q, wherein the alkyl or alkoxy groups are saturatedor unsaturated, linear or branched, unsubstituted or optionallysubstituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl,amide, thioamide, or Q, and the aryl group is optionally substituted byF, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide,or Q; or

one or more of R¹ in combination with R², R² in combination with R³, R³in combination with R⁴, R⁴ in combination with R⁵, R⁶ in combinationwith R⁷, and R⁸ in combination with R⁹, form a 5- to 10-member ring thatis saturated or unsaturated, unsubstituted or optionally substitutedwith one or more of alkyl, aryl, alkenyl, alkynyl, alkoxy, aroxyl,hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl,alkylsulfinyl, arylsulfinyl, (thio)carbonyl, (thio)carboxylic acid,(thio)carboxylic acid ester, nitro, amino, (thio)amide, azido,hydrazino, or (thio)phosphonate, wherein the alkyl or alkoxy groups aresaturated or unsaturated, linear or branched, substituted orunsubstituted, and the aryl group is optionally substituted with F, Cl,Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio,arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl,thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester,unsubstituted or substituted amino, amide, thioamide, or Q;

Q is selected from a carboxyl group (CO₂ ⁻), a carbonate ester (COER¹²),a sulfonate ester (SO₂ER¹²), a sulfoxide (SOR¹²), a sulfone(SO₂CR¹²R¹³R¹⁴), a sulfonamide (SO₂NR¹²R¹³), a phosphate (PO₄ ⁼), aphosphate monoester (PO₃ ⁻ER¹²), a phosphate diester (PO₂ER¹²ER¹³), aphosphonate (PO₃ ⁼) a phosphonate monoester (PO₂ ⁻ER¹²) a phosphonatediester (POER¹²ER¹³), a thiophosphate (PSO₃ ⁼), a thiophosphatemonoester (PSO₂ ⁻ER¹²) a thiophosphate diester (PSOER¹²ER¹³), athiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER¹²) athiophosphonate diester (PSER¹²ER¹³), a phosphonamide(PONR¹²R¹³NR¹⁵R¹⁶), its thioanalogue (PSNR¹²R¹³NR¹⁵R¹⁶), a phosphoramide(PONR¹²R¹³NR¹⁴NR¹⁵R¹⁶), its thioanalogue (PSNR¹²R¹³NR¹⁴NR¹⁵R¹⁶), aphosphoramidite (PO₂R¹⁵NR¹²R¹³) or its thioanalogue (POSR¹⁵NR¹²R¹³),wherein E is independently O or S;

R¹⁰ and R¹¹ are each independently selected from H, a saturated orunsaturated, linear or branched, unsubstituted or further substitutedalkyl group, aryl group, alkylcarbonyl, aryl carbonyl,alkylthiocarbonyl, arylthiocarbonyl, alkoxycarbonyl, aroxycarbonyl,alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl,diarylaminocarbonyl, alkyl(aryl)aminocarbonyl, arylcarboxamido, or Q,the alkyl or alkoxy portions of which are, alkenyl, alkynyl,alkylcarbonyl, amide, thioamide, or Q, or the aryl portions of which areoptionally substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl,alkylcarbonyl, amide, thioamide, or Q; or

at least one of R⁷ in combination with R¹⁰, or R⁹ in combination withR¹¹ forms a 5- to 10-member saturated or unsaturated ring optionallyfurther substituted with one or more saturated or unsaturated, linear orbranched, substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl,saturated or unsaturated, linear or branched, substituted orunsubstituted alkoxy, aroxyl, hydroxyl, F, Cl, Br, I, CN, nitro,alkylsulfonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl,(thio)carbonyl, (thio)carboxylic acid, (thio)carboxylic acid ester,nitro, amino, (thio)amide, azido, hydrazino, or (thio)phosphonate;wherein the aryl group is optionally substituted with one or more of F,Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio,arylthio, nitro, azido, hydrazino, carboxyl, thiocarboxyl, carbonyl,thiocarbonyl, carboxylic acid ester, thiocarboxylic acid ester,unsubstituted or substituted amino, amide, thioamide, or Q;

wherein R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected fromhydrogen, a halogen, an amino group, a saturated or unsaturated, linearor branched, substituted or unsubstituted alkyl group, a saturated orunsaturated, branched or linear, substituted or unsubstituted alkoxygroup, a substituted or unsubstituted aryl group; or R¹² in combinationwith R¹³, R¹⁴ in combination with R¹⁶, R¹² in combination with R¹⁴, R¹²in combination with R¹⁵, R¹³ in combination with R¹⁶, and R¹⁴ incombination with R¹⁵, one or more of which, form a 5- to 10-member ring;and

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ comprises one or more reactive groups Z,selected from isocyanate, isothiocyanate, monochlorotriazine,dichlorotriazine, 4,6-dichloro-1,3,5-triazines, mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,maleimide, haloacetamide, aziridine, sulfonyl halide, carboxylic acid,acid halide, phosphonyl halide, phosphoramidite (PO₂R¹⁵NR¹²R¹³) or itsthioanalogue (POSR¹⁵NR¹²R¹³), hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, azidonitrophenol ester,azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, thiol,amine, hydrazine, hydroxyl, terminal alkene, a terminal alkyne, aplatinum coordinate group and an alkylating agent.

In other embodiments, the disclosure is directed to a dye having thestructure of Formula II:

wherein at least one of R¹ or R² is a nitro group;

X is O, S, or NR¹⁷, wherein R¹⁷ is selected from H, an alkyl group thatis saturated or unsaturated, linear or branched, unsubstituted, orfurther substituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl,alkylcarbonyl, amide, thioamide, or Q, or an aryl group optionallysubstituted by F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl,amide, thioamide, or Q,

Q is selected from a carboxyl group (CO₂ ⁻), a carbonate ester (COER¹²),a sulfonate ester (SO₂ER¹²), a sulfoxide (SOR¹²), a sulfone(SO₂CR¹²R¹³R¹⁴), a sulfonamide (SO₂NR¹²R¹³), a phosphate (PO₄ ⁼), aphosphate monoester (PO₃ ⁻ER¹²), a phosphate diester (PO₂ER¹²ER¹³), aphosphonate (PO₃ ⁼) a phosphonate monoester (PO₂ ⁻ER¹²) a phosphonatediester (POER¹²ER¹³), a thiophosphate (PSO₃ ⁼), a thiophosphatemonoester (PSO₂ ⁻ER¹²) a thiophosphate diester (PSOER¹²ER¹³), athiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER¹²) athiophosphonate diester (PSER¹²ER¹³), a phosphonamide(PONR¹²R¹³NR¹⁵R¹⁶), its thioanalogue (PSNR¹²R¹³NR¹⁵R¹⁶), a phosphoramide(PO₂R¹⁵NR¹²R¹³), its thioanalogue (PSNR¹²R¹³NR¹⁴NR¹⁵R¹⁶), aphosphoramidite (PO₂R¹⁵NR¹²R¹³) or its thioanalogue (POSR¹⁵NR¹²R¹³) andE is independently O or S;

R¹, R², R⁶, R⁷, R⁸, and R⁹ are each independently selected from H, F,Cl, Br, I, CN, nitro, azido, hydroxyl, amino, hydrazino, aryl, aroxyl,alkenyl, alkynyl, alkyl, alkoxy, alkylamino, dialkylamino, arylamino,diarylamino, alkylamino, alkylarylamino, alkanoylamino, alkylthio,alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl,alkyloxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl,arylcarboxamido, or Q, wherein the alkyl or alkoxy groups are saturatedor unsaturated, linear or branched, unsubstituted or further substitutedby F, Cl, Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide,thioamide, or Q, and the aryl group is optionally substituted by F, Cl,Br, I, CN, OH, alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q;

one or more of R¹ in combination with R¹⁷, R⁶ in combination with R⁷,and R⁸ in combination with R⁹ form a saturated or unsaturated 5- to10-member ring optionally substituted by one or more of a saturated orunsaturated, linear or branched, substituted or unsubstituted alkygroup, aryl, alkenyl, alkynyl, a saturated or unsaturated, branched orlinear, substituted or unsubstituted alkoxy group, aroxyl, hydroxyl, F,Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl, alkylsulfinyl,arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid, thiocarboxylicacid, carboxylic acid ester, thiocarboxylic acid ester, nitro, amino,amide, thioamide, azido, hydrazino, phosphonate or thiophosphonate,wherein the aryl group is optionally substituted with F, Cl, Br, I, CN,OH, alkyl, alkenyl, alkynyl, alkoxy, aryoxy, alkylthio, arylthio, nitro,azido, hydrazino, carboxyl, thiocarboxyl, carbonyl, thiocarbonyl,carboxylic acid ester, thiocarboxylic acid ester, unsubstituted orsubstituted amino, amide, thioamide, or Q;

R¹⁰ and R¹¹ are each independently selected from H, alkyl, aryl,alkylcarbonyl, aryl carbonyl, alkylthiocarbonyl, arylthiocarbonyl,alkoxycarbonyl, aroxycarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, alkyl(aryl)aminocarbonyl,arylcarboxamido, or Q, wherein the alkyl group and the alkoxy group areeach independently saturated or unsaturated, linear or branched,unsubstituted or further substituted by F, Cl, Br, I, CN, OH, alkenyl,alkynyl, alkylcarbonyl, amide, thioamide, or Q, and the aryl group isunsubstituted or optionally substituted by F, Cl, Br, I, CN, OH,alkenyl, alkynyl, alkylcarbonyl, amide, thioamide, or Q;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are each independently selected fromhydrogen, a halogen, an amino group, a saturated or unsaturated, linearor branched, substituted or unsubstituted alkyl group, a saturated orunsaturated, linear or branched, substituted or unsubstituted alkoxygroup, a substituted or unsubstituted aryl group; or one or more of R¹²in combination with R¹³, R¹⁴ in combination with R¹⁶, R¹² in combinationwith R¹⁴, R¹² in combination with R¹⁵, R¹³ in combination with R¹⁶, andR¹⁴ in combination with R¹⁵ form a 5- to 10-member ring;

at least one of R⁷ in combination with R¹⁰ , or R⁹ in combination withR¹¹ form a saturated or unsaturated 5- to 10-member ring optionallysubstituted with alkyl, aryl, alkenyl, alkynyl, alkoxy, aroxyl,hydroxyl, F, Cl, Br, I, CN, nitro, alkylsulfonyl, arylsulfonyl,alkylsulfinyl, arylsulfinyl, carbonyl, thiocarbonyl, carboxylic acid,thiocarboxylic acid, carboxylic acid ester, thiocarboxylic acid ester,nitro, amino, amide, thioamide, azido, hydrazino, phosphonate, orthiophosphonate wherein the alky group and the alkoxy group are eachindependently saturated or unsaturated, linear or branched, substitutedor unsubstituted, and wherein the aryl group is unsubstituted orsubstituted with F, Cl, Br, I, CN, OH, alkyl, alkenyl, alkynyl, alkoxy,aryoxy, alkylthio, arylthio, nitro, azido, hydrazino, carboxyl,thiocarboxyl, carbonyl, thiocarbonyl, carboxylic acid ester,thiocarboxylic acid ester, unsubstituted or substituted amino, amide,thioamide, or Q;

R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from hydrogen, ahalogen, an amino group, a saturated or unsaturated, linear or branched,substituted or unsubstituted alkyl group, a saturated or unsaturated,branched or linear, substituted or unsubstituted alkoxy group, or anunsubstituted or substituted aryl group; or one or more of R¹² incombination with R¹³, R¹⁴ in combination with R¹⁶, R¹² in combinationwith R¹⁴, R¹² in combination with R¹⁵, R¹³ in combination with R¹⁶, andR¹⁴ in combination with R¹⁵ form a 5- to 10-member ring; and

at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁴, R¹⁵, R¹⁶ and R¹⁷ comprises one or more reactive groups Z,independently selected from isocyanate, isothiocyanate,monochlorotriazine, dichlorotriazine, 4,6-dichloro-1,3,5-triazines,mono- or di-halogen substituted pyridine, mono- or di-halogensubstituted diazine, maleimide, haloacetamide, aziridine, sulfonylhalide, carboxylic acid, acid halide, phosphonyl halide, phosphoramidite(PO₂R¹⁵NR¹²R¹³) or its thioanalogue (POSR¹⁵NR¹²R¹³), hydroxysuccinimideester, hydroxysulfosuccinimide ester, imido ester, azido, nitrophenolester, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde,thiol, amine, hydrazine, hydroxyl, terminal alkene, a terminal alkyne, aplatinum coordinate group and an alkylating agent.

4.1 Complex Ring Structures

In certain embodiments, certain R groups are joined together to form oneor more fused 5- or 6-membered ring structures. In certain embodiments,the complex rings that are formed between R groups may be unsubstitutedor may be further substituted with any of the R groups describedpreviously to form complex ring structures. Examples of rings andcomplex rings containing the amine group include, but are not limitedto:

4.2 Reactive Groups and Targets

In other embodiments described herein, at least one of the R groups is areactive group thereby allowing the dyes to be attached to a targetmolecule. Examples of reactive groups include, but are not limited to, anucleophilic reactive group, an electrophilic reactive group, a terminalalkene, a terminal alkyne, a platinum coordinate group or an alkylatingagent. The skilled artisan will recognize what types of reactive groupscan be used to attach the dark quencher dyes described herein to aparticular component in the target molecule.

In certain embodiments, the reactive group is an electrophilic reactivegroup. Examples of such electrophilic reactive groups include, but notbe limited to, isocyanate, isothiocynate, monochlorotriazine,dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide,glyoxal and aldehyde groups.

In various embodiments, the reactive group is a nucleophilic reactivegroup. Such nucleophilic reactive groups include, but are not limitedto, reactive thiol, amine and hydroxyl groups. During synthesis of dyes,reactive thiol, amine or hydroxyl groups can be protected, and thereactive groups generated after removal of the protective group. Incertain embodiments, a dye is attached to a terminal alkene or alkynegroup. See, e.g., U.S. Patent Application Serial No. 2003/0225247. Inother embodiments, platinum coordinate groups can be used to attach dyesto a target molecule. See U.S. Pat. No. 5,580,990. In yet otherembodiments, alkyl groups are used to attach dyes to a target molecule.See U.S. Pat. No. 6,593,465.

Examples of target molecules that can be labeled by the monoazo dyesdescribed herein include, but not be limited to, a nucleoside, anucleotide, an oligonucleotide, a polynucleotide, a peptide nucleicacid, a protein, a peptide, an enzyme, an antigen, an antibody, ahormone, a hormone receptor, a cellular receptor, a lymphokine, acytokine, a hapten, a lectin, avidin, strepavidin, digoxygenin, acarbohydrate, an oligosaccharide, a polysaccharide, a lipid, liposomes,a glycolipid, a viral particle, a viral component, a bacterial cell, abacterial component, a eukaryotic cell, a eukaryotic cell component, anatural drug, a synthetic drug, a glass particle, a glass surface,natural polymers, synthetic polymers, a plastic particle, a plasticsurface, a silicaceous particle, a silicaceous surface, an organicmolecule, other dyes and derivatives thereof.

In certain embodiments, the nucleoside, nucleotide, oligonucleotide, orpolynucleotide comprises one or more ribonucleoside moieties,ribonucleotide moieties, deoxyribonucleoside moieties,deoxyribonucleotide moieties, modified ribonucleosides, modifiedribonucleotides, modified deoxyribonucleosides, modifieddeoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotideanalogues, and any combination thereof.

As disclosed above, in certain embodiments, the monoazo dyes describedherein can have other dyes as targets, thereby creating composite dyesin which two or more dyes are covalently attached. In variousembodiments, the composite dyes have unique properties that are notpresent in either dye alone. For example, in certain embodiments, if oneof the dyes described herein is covalently joined to another dye suchthat it creates an extended conjugation system, the spectralcharacteristics of the dye pair may be different than the spectralcharacteristics of either dye component alone. In other embodiments, theconjugation systems of the joined dyes do not overlap but the proximityallows an internal energy transfer to take place, thereby extending theStokes shift. See, e.g., U.S. Pat. Nos. 5,401,847; 6,008,373 B1 and5,800,996. In various embodiments, other properties may also be enhancedby covalently joining two or more dyes. See, e.g., U.S. PatentApplication Publication No. 2003/0225247 (two ethidium bromide moleculesjoined together generates a dye that has enhanced binding to nucleicacids). In certain embodiments, composite dyes exhibit enhanced bindingand energy transfer. See, e.g., U.S. Pat. No. 5,646,264. In particularembodiments, composite dyes include not only two dyes, but can compriseoligomeric or polymeric dyes. In certain embodiments, the composite dyesdescribed herein comprise multimers of same dye. In other embodiments,the composite dyes comprise multimers of different dyes. The skilledartisan will appreciate that the identities of the dyes included inmultimers are dependent on the desired properties of the dye multimers.

Selected embodiments of the monoazo dyes described herein include, butare not limited to, the illustrated dyes:

4.3 Methods of Use

In various embodiments, the dyes described herein are attached to atarget-specific moiety. In these embodiments, binding between thetarget-specific moiety and its corresponding target is monitored byessentially determining the presence or amount of dye that is bound tothe target. Well-known examples of such assays include hybridizationsbetween complementary nucleic acids as well as binding that take placebetween antibodies and their corresponding antigens. Other binding pairsof interest include, but are not limited to, ligand/receptor,hormone/hormone receptor, carbohydrate/lectin and enzyme/substratepairs. In certain embodiments, assays are carried out in which a firstcomponent of the binding pair is fixed to a solid support, and a secondcomponent of the binding pair is in solution. Accordingly, in theseembodiments, by binding to the first component fixed to the support, thesecond component also is attached to the support. In particularembodiments, the binding pairs described herein are used in microarrayassays, where labeled analytes become bound to discrete sites on themicroarray. In other particular embodiments, binding pairs describedherein are used in homogeneous probe-dependent assays. Examples of suchmethods include, but are not limited to, energy transfer betweenadjacent probes (U.S. Pat. No. 4,868,103), the Taqman exonuclease assay(U.S. Pat. Nos. 5,538,848 and 5,210,015), molecular beacon assays (U.S.Pat. Nos. 5,118,801 and 5,925,517) and various real time assays (U.S.patent application Ser. No. 10/096,076).

In various embodiments, the dyes described herein can be used asquenchers in energy transfer systems for detection and/or quantificationof proteins or nucleic acids. In particular embodiments, energy transferis detected by an increase in signal from a Fluorescence ResonanceEnergy Transfer (FRET) acceptor as it absorbs energy from a FRET donor.In other systems, energy transfer can be detected by a loss of signalfrom a donor as it transfers energy to an energy acceptor. See, e.g.,Livak et al. (1995) PCR Methods and Applications 4:357-362 (earlyversions of TaqMan® using Fluorescein as a donor and TAMRA as anacceptor at opposite ends of a probe provided a quenched probe systemuseful for detecting PCR product); Gibson et al. (1996) Genome Research6; 995-1001; Wittwer et al. (1997) Biotechniques 22:130-138 (describingTaqMan® or molecular beacon probe assays in which the loss of energytransfer generates a signal or signal is generated by the creation ofFRET).

In certain embodiments, the compositions disclosed herein can be used inreal-time PCR reactions that utilize a variety of differentconformations. See, e.g., Arya et al. (2005) Expert Rev Mol Diagn.5:209-219; Marras et al. (2005) Clinica Chemica Acta 363:48-60; Wong andMedrano (2005) Biotechniques 39:75-85; and U.S. Pat. No. 8,241,179(real-time PCR technique in which each primer used for PCR-basedamplification has an energy transfer element, and primer locations aredesigned such that an amplicon has two energy transfer elements insufficient proximity that energy is transferred from one extended primerto the energy transfer acceptor on the primer of the other strand).Another example of a real-time PCR methods in which the quenchersdescribed herein can be used are described in U.S. Pat. No. 8,241,179“Process for Quantitative or Qualitative Detection of Single-strandedNucleic Acids”. In this system, multiplex amplification can be carriedout using a variety of different fluors where the least complexity isobtained by using either multiple acceptors and a single donor or asingle acceptor (such as a quencher described herein) and multipledonors.

In other embodiments, the dyes described herein can be used in methodsother than classical PCR methods, such as isothermal amplificationsystems that generate nucleic acid products and use primers and/orprobes labeled with quenchers. See, e.g., Gill and Ghaemi (2008)Nucleosides, Nucleotides and Nucleic Acids 27:224-243. In particularembodiments, the quencher dyes described herein are adapted for use invarious immunoassay formats for protein quantification. See, e.g.,Niemeyer et al. (2005) TRENDS in Biotechnology 23:208-216, and Gullberget al. (2004) Proc. Nat Acad Sci (USA) 101:8420-8424.

In a particular embodiment, the dark quencher dyes described herein canbe used in methods for detecting qualitatively or quantitatively thepresence of a single-stranded nucleic acid of interest in a samplecomprising the steps of (a) providing (i) a composition of mattercomprising at least two parts: a first part comprising at least onefirst nucleic acid primer that comprises (A) at least one first energytransfer element; and (B) a nucleic acid sequence that is complementaryto a nucleotide sequence in at least a portion of the nucleic acid ofinterest; and a second part comprising at least one second nucleic acidprimer that comprises: (A′) at least one second energy transfer element;and (B′) a nucleic acid sequence that is identical to a nucleotidesequence in at least a portion of the nucleic acid of interest; whereinthe first nucleic acid primer does not comprise the second energytransfer element, and wherein the second nucleic acid primer does notcomprise the first energy transfer element, the first energy transferelement is an energy transfer donor and the second energy transferelement is a quencher, or the first energy transfer element is anquencher and the second energy transfer element is an energy transferdonor, and neither the first nucleic acid primer nor the second nucleicacid primer is fixed or immobilized to a solid support; (ii) a samplesuspected of containing the nucleic acid of interest; and (iii) reagentsfor carrying out nucleic acid strand extension; (b) forming a reactionmixture comprising (i), (ii) and (iii) above; (c) contacting underhybridization conditions the first nucleic acid primer with one strandof the nucleic acid of interest and contacting under hybridizationconditions the second nucleic acid primer with the complementary strandof the nucleic acid of interest, if present; (d) extending the firstnucleic acid primer and the second nucleic acid primer to form a firstprimer-extended nucleic acid sequence and a second primer-extendednucleic acid sequence if the complementary strand is present; (e)separating the first primer-extended nucleic acid sequence from thenucleic acid of interest and separating the second primer-extendednucleic acid sequence from the complementary strand of the nucleic acidof interest if present; (f) contacting under hybridization conditionsthe first nucleic acid primer with the nucleic acid of interest or thesecond primer-extended nucleic acid sequence from step (e), andcontacting under hybridization conditions the second nucleic acid primerwith the first primer-extended nucleic acid sequence from step (e); and(g) detecting the presence or quantity of the nucleic acid of interestby detecting energy transfer between the first and second energytransfer elements by means of loss of signal from the first energytransfer donors.

5. EXAMPLES

This section will describe the various different working examples thatwill be used to highlight the features of the invention(s).

5.1 Example 1. Synthesis of7-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Compound 1)

m-Anisidine (26 ml, 0.23 mol) was added slowly to acetic acid (2.6 ml)with stirring, followed by the addition of mesityl oxide (27 ml, 0.23mol) to the solution. The mixture was stirred at room temperatureovernight. Concentrated hydrobromic acid (50 ml) was added. The mixturewas stirred for an additional hour. The solid formed was collected byfiltration and then washed with acetone (3×50 mL). The resulting solidwas dissolved in water (100 ml) and neutralized to pH 7 with 10N aqueoussodium hydroxide. The precipitate was extracted with chloroform (3×50mL) and dry over anhydrous sodium sulfate. After filtering off sodiumsulfate, the solvent was evaporated under vacuum to give crude product.The crude product was recrystallized with hexanes to give compound 1 asyellowish solid (15.5 g, 33% yield). The structure of compound 1 isgiven below:

5.2 Example 2. Synthesis of4-(7-methoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoic acid (Compound 3)

Calcium carbonate (6.01 g, 60 mmols) and ethyl 4-bromobutyrate (9.75 g,50 mmols) were added to a solution of compound 17-methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (8.12 g, 40 mmols) inanhydrous DMF (100 ml). The mixture was stirred at 120° C. for 3 days(reaction was monitored by TLC: hexane/ethyl acetate, 4/1). The solventwas removed under vacuum. The residue was redissolved in ethyl acetate(200 mL) and filtered through celite. The solvent was removed undervacuum to give crude ester 2.

The crude ester 2 was dissolved in methanol (150 mL) and water (20 mL).Potassium hydroxide (6 g) was added. The mixture was stirred at roomtemperature for one day. The solvent was removed under vacuum. Water(200 mL) and ethyl ether (150 mL) were added to the residue. The waterlayer was extracted with ethyl ether (2×150 mL) and then neutralized topH 3-4 with 6N HCl. The mixture was extracted with ethyl acetate (3×150mL). The combined ethyl acetate layer was washed with water (2×150 mL)and brine (200 mL) and then dried with anhydrous sodium sulfate. Thesolvent was removed under vacuum to give compound 3 as viscous oil (6.7g, 58%). The structure of compound 3 is given below:

5.3 Example 3. Synthesis of 4-nitrobenzenediazonium tetrafluoroborate(Compound 4)

A suspension of 4-nitroaniline (6.216 g, 45 mmols) in 4N HCl (50 mL) wasstirred and cooled with an ice bath for 15 min. A solution of sodiumnitrite (3.42 g, 49.5 mmols) in water (20 mL) was added slowly. Afterthe addition, the mixture was stirred at this temperature for 30 min. Asolution of lithium tetrafluoroborate (5.9 g, 63 mmols) in water (20 mL)was added. The solid precipitate was collected by filtration and washedwith water (2×25 mL), methanol (25 mL) and ether (2×25 mL). Theprecipitate was dried under vacuum overnight to give compound 4 asoff-white solid (7.518 g, 71%). The structure of compound 4 is givenbelow:

5.4 Example 4. Synthesis of(E)-4-(7-methoxy-2,2,4-trimethyl-6-((4-nitrophenyl)diazenyl)quinolin-1(2H)-yl)butanoicacid (Compound 5)

Compound 4 (2.39 g, 10.1 mmols) was added in small batches to a solutionof compound 3 (2.43 g, 8.40 mmols) in pyridine (50 mL) at roomtemperature with stirring. The mixture was stirred at room temperaturefor 3 hours (monitor the reaction by TLC: 5% methanol indichloromethane). The solvent was removed under vacuum. The residue wasredissolved in dichloromethane (200 mL) and water (200 mL). Thedichloromethane layer was washed with water (3×200 mL) and dried withanhydrous sodium sulfate. The solvent was removed under vacuum. Theresidue was purified by flash chromatography (gradient: 0-5% methanol indichloromethane) to afford compound 5 as dark solid (1.70 g, 46%). Thestructure of compound 5 is given below:

5.5 Example 5. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(7-methoxy-2,2,4-trimethyl-6-((4-nitrophenyl)diazenyl)quinolin-1(2H)-yl)butanoate(Compound 6)Method 1: In Situ Activation

A 20 mM solution of compound 5 in DMF (50 μl) was mixed with a 100 mMsolution of TSTU (2-succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate) in DMF (11 μl, 1.1 equivalent) and a 300 mM solutionof DIPEA (diisopropylethylamine) in DMF (7.3 μl, 2.2 equivalent). Themixture was kept at room temperature for 1 hour (monitor the reactionwith TLC: hexanes/ethyl acetate, 2/1). The solution was used directlyfor conjugation with biomolecules.

Method 2: Synthesis of Compound 6 (Isolated)

TSTU (2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate, 412mg, 1.37 mmols) and DIPEA (diisopropylethylamine, 390.3 μL, 2.28 mmols)was added to a solution of compound 5 (500 mg, 1.14 mmols) in DMF (20mL). The mixture was stirred at room temperature for 3 hours (monitorthe reaction by TLC: hexanes/ethyl acetate, 2/1). The solvent wasevaporated to dryness under vacuum. The residue was dissolved indichloromethane (200 mL) and washed with water (3×200 mL) and brine (200mL). The solution was dried with anhydrous sodium sulfate, then filteredand evaporated to dryness to give product as a dark solid (136.5 mg,22%). The structure of compound 6 is given below:

5.6 Example 6. Synthesis of4-(7-methoxy-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoic acid(Compound 7)

Palladium on carbon (10% w/w, 0.1 g) was added to a solution of compound3 (0.9 g) in methanol (50 mL). The mixture was shaken on a hydrogenationapparatus under 50 psi of hydrogen. After the reaction was complete (asmonitored by TLC: hexanes/ethyl acetate, 4/1), the mixture was filteredthrough a pad of celite. The solvent was removed under vacuum to providecompound 7 as a dark green solid (0.8 g, 88%). The structure of compound7 is given below:

5.7 Example 7. Synthesis of(E)-4-(7-methoxy-2,2,4-trimethyl-6-((4-nitrophenyl)diazenyl)-3,4-dihydroquinolin-1(2H)-yl)butanoicacid (Compound 8)

Compound 8 (25.5 mg, 58%) was made from compound 7 (29.1 mg) andcompound 4 (23.7 mg) following the procedure in Example 4. The structureof compound 8 is given below:

5.8 Example 8. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(7-methoxy-2,2,4-trimethyl-6-((4-nitrophenyl)diazenyl)-3,4-dihydroquinolin-1(2H)-yl)butanoate(Compound 9)

Compound 9 was made from compound 8 following the procedure in Example5. The structure of compound 9 is given below given below:

5.9 Example 9. Synthesis of(E)-4-(7-methoxy-6-((2-methoxy-4-nitrophenyl)diazenyl)-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoicacid (Compound 10)

Compound 10 (98.2 mg, 36%) was made from compound 7 (171 mg) and FastRed B salt (274.3 mg) following the procedure in Example 4. Thestructure of Compound 10 is given below:

5.10 Example 10. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(7-methoxy-6-((2-methoxy-4-nitrophenyl)diazenyl)-2,2,4-trimethyl-3,4-dihydroquinolin-1(2H)-yl)butanoate(Compound 11)

Compound 11 was made from compound 10 following the procedure in Example5. The structure of compound 11 is given below:

5.11 Example 11. Synthesis of 2-chloro-4-nitrobenzenediazoniumtetrafluoroborate (Compound 12)

Compound 12 (1.52 g, 12%) was prepared from 2-chloro-4-nitroaniline(7.77 g) following the procedure in Example 3. The structure of Compound12 is given below:

5.12 Example 12. Synthesis of(E)-4-(6-((2-chloro-4-nitrophenyl)diazenyl)-7-methoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoicacid (Compound 13)

Compound 13 (7.2 mg, 15%) was prepared from compound 12 (27.1 mg) andcompound 3 (28.9 mg) following the procedure in Example 4. The structureof Compound 13 is given below:

5.13 Example 13. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(6-((2-chloro-4-nitrophenyl)diazenyl)-7-methoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoate(Compound 14)

Compound 14 was prepared from compound 13 following the procedure inExample 5. The structure of Compound 14 is given below:

5.14 Example 14. Synthesis of(E)-4-(7-methoxy-2,2,4-trimethyl-6-((5-nitrothiazol-2-yl)diazenyl)-3,4-dihydroquinolin-1(2H)-yl)butanoicacid (Compound 15)

Sodium nitrite (58.1 mg) was added slowly to concentrated sulfuric acid(0.42 mL) with shaking and cooling. The mixture was maintained at 0° C.for 30 min, then added to a solution of 2-amino-5-nitrobenzothiazole(120 mg) in acetic acid (1.5 mL) at room temperature. After theresulting mixture was stirred at 0° C. for 1 hour, a solution ofcompound 7 (200 mg) in acetic acid (1.9 mL) was added. The mixture wasstirred at room temperature for 3 hours, and then poured into ice-water(15 mL). The resultant precipitate was extracted with ethyl acetate(3×15 mL) and the combined ethyl acetate layer was washed with water(3×30 mL) and brine (30 mL). After drying with anhydrous sodium sulfate,the solvent was removed under vacuum. The residue was purified by flashchromatography (gradient: 0% to 5% of methanol in dichloromethane) togive compound 15 as dark solid (40.1 mg, 11%). The structure of compound15 is given below:

5.15 Example 15. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(7-methoxy-2,2,4-trimethyl-6-((5-nitrothiazol-2-yl)diazenyl)-3,4-dihydroquinolin-1(2H)-yl)butanoate(Compound 16)

Compound 16 was prepared from compound 15 following the procedure as inExample 5. The structure of compound 16 is given below:

5.16 Example 16. Synthesis of(E)-4-(7-methoxy-2,2,4-trimethyl-6-((5-nitrothiazol-2-yl)diazenyl)quinolin-1(2H)-yl)butanoicacid (Compound 17)

Compound 17 (20.3 mg, 23%) was prepared from compound 3 (57.9 mg)following the procedure as in Example 14. The structure of compound 17is given below:

5.17 Example 17. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(7-methoxy-2,2,4-trimethyl-6-((5-nitrothiazol-2-yl)diazenyl)quinolin-1(2H)-yl)butanoate(Compound 18)

Compound 18 was prepared from compound 17 following the procedure as inExample 5. The structure of compound 18 is given below:

5.18 Example 18. Synthesis of5,7-dimethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Compound 19)

Compound 19 (19.3 g, 83%) was prepared from 3,5-dimethoxyaniline (15.3mg) following the procedure in Example 1. The structure of compound 19is given below:

5.19 Example 19. Synthesis of5,7-dimethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Compound 21)

Compound 21 (11.2g, 70%) was prepared through compound 20 from compound19 (11.67 g) following the procedure as in Example 2. The structures ofcompound 20 and compound 21 are given below:

5.20 Example 20. Synthesis of(E)-4-(6-((2-cyano-4-nitrophenyl)diazenyl)-7-methoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoicacid (Compound 22)

Compound 22 (265.3mg, 33%) was prepared from 2-amino-5-nitrobenzonitrile(338 mg) and compound 3 (500 mg) following the procedure as in Example14. The structure of compound 22 is given below:

5.21 Example 21. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(6-((2-cyano-4-nitrophenyl)diazenyl)-7-methoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoate(Compound 23)

Compound 23 was prepared from compound 22 following the procedure as inExample 5. The structure of compound 23 is given below:

5.22 Example 22. Synthesis of(E)-4-(6-((2-cyano-4-nitrophenyl)diazenyl)-5,7-dimethoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoicacid (Compound 24)

Compound 24 (22.1 mg, 22%) was prepared from compound 21 (63.9 mg) and2-amino-5-nitrobenzonitrile (32.5 mg) following the procedure in Example14. The structure of compound 24 is given below:

5.23 Example 23. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(6-((2-cyano-4-nitrophenyl)diazenyl)-5,7-dimethoxy-2,2,4-trimethylquinolin-1(2H)-yl)butanoate(Compound 25)

Compound 25 was prepared from compound 24 following the procedure inExample 5. The structure of compound 25 is given below:

5.24 Example 24. Synthesis of Compound 26

Compound 26 was prepared from compound 1 and 4-bromobutanol followingthe procedure in Example 2. The structure of compound 26 is given below:

5.25 Example 25. Synthesis of Compound 27

Compound 27 was prepared from compound 4 and compound 26 following theprocedure in Example 4. The structure of compound 27 is given below:

5.26 Example 26. Synthesis of Compound 28

A solution of 2-cyanoethyl tetraisopropylphosphorodiamidite (190 mg,0.64 mmol) in dichloromethane (5 mL) was added to a solution of compound27 (255 mg, 0.6 mmol) and diisopropylammonium tetrazolide (52 mg, 0.3mmol) in dichloromethane (10 mL) at room temperature. After the mixturewas stirred at room temperature overnight, it was washed with saturatedsodium bicarbonate (15 mL), water (2×15 mL) and brine (15 mL). Thesolution was dried with anhydrous sodium sulfate and then evaporatedunder vacuum. The resulting crude product was purified by flashchromatography on silica gel. Compound 28 was obtained as a dark coloredpowder (232 mg, 62%). The structure of compound 28 is given below:

5.27 Example 27. Synthesis of Compound 29

Compound 29 was prepared from 2-amino-5-nitrobenzonitrile and compound26 following the procedure in Example 20. The structure of compound 29is given below:

5.28 Example 28. Synthesis of Compound 30

Compound 30 was prepared from compound 29 following the procedure inExample 26. The structure of compound 30 is given below:

5.29 Example 29. Synthesis of Compound 31

To a solution of DMT protected 5-allylamine-dU (Enzo Life Sciences,Inc., 58.6 mg, 0.1 mmol) in acetonitrile (10 mL) was added a solution ofcompound 6 (53.6 mg, 0.1 mmol) in acetonitrile (5 mL). The mixture wasstirred at room temperature overnight and then solvent was evaporatedunder vacuum. The residue was dissolved in dichloromethane (50 mL). Itwas washed with water (3×20 mL), brine (40 mL) and then dried withanhydrous sodium sulfate. The solvent was removed under vacuum. Thecrude product was purified by flash chromatography to provide compound31 as a dark solid (85.9 mg, 84%). The structure of compound 31 is givenbelow:

5.30 Example 30. Synthesis of Compound 32

Compound 32 was prepared from compound 31 following procedure in Example26. The structure of compound 32 is given below:

5.31 Example 31. Synthesis of Compound 33

Compound 33 was prepared from compound 23 following the procedure inExample 29. The structure of compound 33 is given below:

5.32 Example 32. Synthesis of Compound 34

Compound 34 was prepared from compound 33 following the procedure inExample 26. The structure of compound 34 is given below:

5.33 Example 33. General Procedure for Conjugation to Oligonucleotide(with Compound 5 as an Example)

Compound 5 (2 μmol) was dissolved in amine-free DMF (140 μl), followedby the addition of 2-succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate (2.4 μmols) and diisopropylethylamine (4.4 μmols). Themixture was stirred at room temperature for 30 min, and then added to asolution of oligonucleotide containing an amine linker (80 nmols) in 0.9M sodium borate buffer (320 μL, pH 8.5). The combined mixture wasstirred at room temperature for 16 h. Solvents were removed under vacuumand the residue pellet was purified by HPLC using a gradient oftriethylammoniumacetate (0.1 M, pH 6.9) and acetonitrile as elutingsolvents. The fractions containing pure conjugates were combined,evaporated, and co-evaporated with water to remove excessive salt. Thefinal blue pellet was dissolved in water and stored at −20° C. untilfurther use.

5.34 Example 34. General Procedure of Conjugation with Streptavidin(with Compound 5 as an Example)

Compound 5 (175 nmol) was dissolved in amine-free DMF (35 μl), followedby the addition of 2-succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate (192.5 nmol) and diisopropylethylamine (350 nmol). Themixture was stirred at room temperature for 60 min, and then added insmall aliquots to a solution of streptavidin (17.5 nmol) in 100 mMcarbonate/bicarbonate buffer (350 μL). The mixture was stirred at roomtemperature for 1 hour. The mixture was loaded to the top of a NAP™ 25gel filtration column and eluted with 1×PBS buffer. The fractionscontaining the dye-streptavidin conjugate were combined. BSA solution(50 mM, 43.2 μL) and 20% NaN₃ solution (7.5 μL) were added. The mixturewas stored at 4° C.

5.35 Example 35. General Procedure of Conjugation to Amine ModifiedNucleotides Using Compound 5 as an Example

Compound 5 (12 μmol) was dissolved in amine-free DMF (840 μl), followedby the addition of 2-succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate (14.4 μmol) and diisopropylethylamine (26.4 μmol). Themixture was stirred at room temperature for 60 min, and then added to asolution of allylamine-dUTP (2′-deoxyuridine, 5′-triphosphate, 10 μmol)in 0.1 M sodium borate buffer (840 μL, pH 8.5). The mixture was stirredat room temperature for 16 h. Pure product (3.8 μmol, 38% yield) wasobtained by ion exchange chromatography. The structure of theallylamine-dUTP and compound 5 conjugate is given below:

The conjugates of compound 5 with ATP, GTP, CTP, TTP, UTP, dATP, dGTP,dCTP, dTTP, ddUTP, ddATP and ddCTP were prepared through similarprocedures using respective modified nucleotides containing aminogroups.

5.36 Example 36. qPCR Studies with Compound 6 and 23

Compounds 6 and 23 were shown to function in a qPCR assay (Enzo LifeSciences Inc., Farmingdale, N.Y., U.S. Pat. No. 8,247,179). Thefluorescent dye labeled oligo YpF574 (5′-CAGACGA-ATTCATTTGCCTGAAGTAG-3′)[SEQ ID NO:1] labeled on the third base from the 3′ end was used withcompound 6 and 23 labeled oligo YpR600(5′-ATTCATGAGTTGAAATCACT-GGTT-CCTC-3′) [SEQ ID NO:2] labeled on thepenultimate base to amplify the target sequenceCAGACGAATTCGATTTGCCTGAAGTAGAGGAACCAGTGATTTCAACTCATGAAT [SEQ ID NO:3], oran unmatched sequence. The oligos were purchased with an amino group onthe 5 position of the thymine base for labeling with various dyes. TheNHS ester of the dye (Fluorescein, Red598s or Cy5) or compound 6 and 23were added in a 20-fold excess to the oligo in 50 mM sodium carbonate,pH 9.6, 50% dimethyl formamide, and incubated with shaking at 22° C. for2 hours. The reaction mixture was then dried in a SpeedVac vacuumconcentrator. The dried oligos were resuspended in 400 μl water and 40μl of 3 M sodium acetate, pH 5.3 was added to this, followed by 1 ml ofice cold ethanol. The combined mixture was then chilled at −80° C. for 1hour, and then centrifuged at 16,000×g for 45 minutes. The supernatantcontaining unincorporated dye was removed using vacuum aspiration. 400μl of 70% ethanol was added, and the tube was again centrifuged for 30minutes at 16,000×g. The supernatant was removed using vacuumaspiration. After removal of all ethanol, the oligos were resuspended in50 μl of water. The oligos were HPLC purified prior to use in PCR usingstandard methods known to those in the field.

PCR was performed with 0.55 μM of the YpF574 and YpR600 in buffer (50 mMHEPES, pH 7.6, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2.5 mM MgCl₂, 0.002% Tween20,5% sucrose, 200 mM Betaine, 160 mM 1,2-propanediol, 200 mM each of thedNTPs) with 1.5 units of Ampigene HS Taq DNA Polymerase (ENZO,Farmingdale, N.Y.). About 10 to 20 copies of the target DNA mixed with 1μg/ml single-stranded Salmon Sperm DNA (SIGMA, St. Louis, Mo.) and thenadded to the reaction, and the enzyme was activated at 95° C. for 5minutes followed by 55 cycles of 95° C. 15 seconds, 68° 40 seconds.Fluorescence measurement was recorded after the 68° step. The reactionwas performed in either a Qiagen Rotorgene (for Cy5) or RocheLightCycler 2 (for Fluorescein and ENZO Red598). FIGS. 7-9 show thequenching functionality of compounds 6 and 23 with various dyes.

5.37 Example 37. Analysis of High Risk HPV+ Patient Pap Smears for E6/E7Viral mRNA Using Compound 6

A molecular beacon with a hairpin structure with 6-FAM on the 5′ end andcompound 6 on the 3′ end was used in this study. This construct wastargeted to the mRNA of high risk HPV E6/E7 (each probe at aconcentration of 8 nM).

Pap smear samples fixed in ThinPrep solution were spun down, supernatantwas aspirated, and cells were resuspended in PBS containing 5%formaldehyde. Cells were incubated for 30 minutes, then washed 3 timeswith PBS. Cells were resuspended in hybridization buffer (2% TritonX-100 in 1×SSC) containing a cocktail of molecular beacons. Cells wereincubated in the dark at 65° C. for 1 hour to induce hybridization tothe target sequences, then at 4° C. for 30 minutes to ensure unboundprobes returned to their hairpin structure. Cells were then run in aFACSCalibur flow cytometer to measure bound beacons. FIG. 10 shows atypical E6/E7 negative Pap smear sample and FIG. 11 a typical E6/E7positive Pap smear sample.

What is claimed is:
 1. A compound selected from the group consisting of


2. The compound of claim 1 having the structure


3. The compound of claim 1 having the structure